Review of Literature: Leishmaniasis

Transcription

1 Review of Literature: Leishmaniasis

2 Leishmaniasis Parasitic diseases were always a heavy burden for humanity. Protozoan parasites are responsible for several important diseases that threaten the lives of nearly one quarter of the human population worldwide. Leishmaniasis is a disease complex, caused by haemoflagellate obligate intracellular protozoa belonging to the genus Leishmania, family Trypanosomatidae of the order Kinetoplastida. Leishmania parasites were previously observed by David D. Cuningham in 1885 and Peter Borovsky in These parasites were mistaken for other protozoa, but later named as Helcosoma tropica by James Wright in Luhe in 1906 renamed it as Leishmania tropica. Similarly, the causative agent of visceral leishmaniasis was first named as Pyroplasma donovani which was rechristened as Leishmania donovani by Ross in 1903 after its discoverers Leishman (1900) from London and Donovan (1903) from Madras who reported the organism independently (Herwaldt, 1999). There are more than 20 different protozoan species of the genus, Leishmania that are pathogenic for humans and transmitted by an insect vector, the phlebotomine sand fly. The leishmaniasis has a long history of its origin. Designs on pre-colombian pottery and the existence of thousand year old skulls prove that the disease has been present in the Americans for a long time (Manson-Bahr, 1996). It has also been present in Africa and India since the mid eighteenth century (Berhe et al., 1999). It ranks second only to malaria, causing considerable morbidity and mortality in tropical and subtropical regions of the world. The control of leishmaniasis remains a serious problem and with ever increasing cases world wide, it has become a major focus of concern and a serious third World problem afflicting the poorer sections of the society (WHO, 2002) Taxonomy The classification of Leishmania was initially based on ecobiological criteria such as vectors, geographical distribution, tropism, antigenic properties and clinical manifestations

3 (Marsden and Lumsden, 1971; Bray, 1974; Pratt and David, 1981; Ryan et al., 1990). However, biochemical and molecular analysis showed that pathological and geographical criteria were often inadequate and thus, other criteria such as the patterns of polymorphism exhibited by kinetoplastic DNA (k- DNA) markers, proteins or antigens came to be used to classify Leishmania (Arnot and Barker, 1981; Miles et al., 1981; de Ibarra et ai., 1982; Handman and Curtis, 1982; Wirth and Pratt, 1982; Brainard et al., 1986; Le Blancq et ai., 1986; Travi et al., 2002). A modem scheme of classification of Leishmania is shown in Fig All members of the genus Leishmania Ross, 1903 are parasites of mammals. The two subgenera, Leishmania and Viannia, are separated on the basis of their location in the vector's intestine (Ryan et a/., 1990). Rioux et a/. (1990) used isoenzyme analysis to define species complexes within the subgenera. Initially, species classification was based Subkingdom Order Protozoa Kinctoplaslida Family Genus SubReaus Compln Speties t:,iljujto l.eptn1noiws IllI"pcUIIPH)I'tID mtn'oci'iih,,}ia l.euhmon,o I I lreti#mtunia '" Joj''OI1i I. "Tica I. jor L, artopl<q I. mixi"""" /.",arri!llmldl I. kin/ekl I. IIKIJ'" I" O<lhwpi<u I" amllt_luis L "'.. gosl I. itfanl.1fi'! /., d01wwini I. lropica I Sauroielsitmmua I PJrytomoltOs I Vlannla I I I I 1" _ 1 I. brazllielufs T. ~'U)"""/UI$ I.. IlIJtfJi T. loi,tf(lfi/. I. braj"",,, I.. Jm,,.,,, t_lfji '- lalom 1 I."gat:nhami r~-"""-...l--,...,-~.. ~- L.. Pf(lll'IanQ I" KUJ UII('Il'f.IS I~ lm',n,'qijq I Nun paihllxcmc,'!(jf I I...'ihtnt1! hrmaallf I. pi/""", i Old World I I j arah,ca ", r:t.'ljczur e,,,sij L Kt'rlnlll L filrallmu i I.. Itll'tU1it'a I i N~' World i t I.. awilje.u i I~ ('lintw! i '- w,,",' i L.,,,.0,~~"-'~/ J, I Nofinol! t dtlt.fljicajion I I J.. "u/(}mbtt!nj/j - L_!:..~!'~~~~~J Fig.I.1: Taxonomy of Leishmania (Based on the scheme published by the World Health Organization [WHO, 1990]). on various extrinsic criteria such as clinical, geographical and biological characteristicsfor example, L. guyanensis (isolated in Guyana), L. peruviana (isolated in Peru), L. infantum (isolated from a child in Tunisia) and L. gerbilli (isolated from gerbils). Since the 1970s, intrinsic criteria such as immunological, biochemical and genetic data have been used to define species of Leishmania. Use of these molecular techniques led to the publication of a taxonomic scheme by the World Health Organization (WHO, 1990). New methods of detection, isolation and genetic identification resulted in a massive increase in the number of species described. Today, 30 species are known and approximately 20 are 4

4 pathogenic for humans. These species generally present different epidemiological and clinical characteristics related to different genetic and phenotypic profiles. The validity of the classification scheme, considered by some workers as too arbitrary, has been questioned several times. Debate has centered on L. panamensis, L. peruviana, L. chagasi, L. infantum, L. archibaldi, L. garnhami, L. pijanoi, L. venezuelensis and L. forattinii (Mauricio et al., 2000; Cupolillo et al., 2001; Sharma et al., 2005). Different studies have already clarified the status of some of these species; for example, L. chagasi is accepted as a synonym of L. infantum (Mauricio et al., 2000) and L. peruviana has been validated as an independent species (Banuls et ai., 2000). The other species listed above are still under discussion Geographical distribution Leishmaniasis has been reported in 88 countries in five continents-africa, Asia, Europe, North America and South America (22 in the New World and 66 in the Old World) (Desjeux, 2001), 16 are developed countries, 72 are developing, and 13 of them are among the least developed (WHO, 2005). Approximately, 350 million individuals are at risk of this disease and 20 million people are infected worldwide, and an estimated 2.0 million new cases occur each year (Leishmaniasis control, leishmaniasis.htm, update 2007) with an incidence of 1.5 million cases per annum of the disfiguring cutaneous leishmaniasis (CL) and 0.5 million cases per annum of the potentially fatal visceral leishmaniasis (VL) (Ashford et al., 1992). However, with increasing travel to and from endemic regions more and more patients with leishmaniasis are seen by physicians in western countries (Herwaldt, 1999; Murray et al., 2000; Guerin et al., 2002). The relevance of this parasitic disease is further stressed out by the rise of LeishmanialHlV coinfection in many parts of the world including European countries such as Spain, Italy, France and Portugal where up to 9% of the AIDS patients suffer from visceral leishmaniasis (Berhe et ai., 1999). Over 90% of the global total of VL cases occurs in five countries; India, Bangladesh, Nepal, Sudan and Brazil (Herwaldt, 1999) and 90% of CL cases occur in seven countries: Afghanistan, Algeria, Brazil, Iran, Peru, Saudi Arabia and Syria. During the past decade there have been epidemics of VL in Sudan (Ashford et al., 1992; Jacquet et ai., 2006), northeast Brazil (Costa et al., 1990), Bangladesh and the states of Patna and Bengal in India (Bolognesi et at., 1999). Leishmaniasis is now an emerging zoonosis in the United States (Enserink, 2000; McHugh et al., 2003; Rosypal et al., 2003) and US soldiers and 5

5 Chapter I : Review of literature peace keeping corps currently in the Middle East are experiencing a large outbreak of leishmaniasis with more than 500 parasitological confirmed cases (CDC, 2004) Leishmaniasis in the Indian subcontinent Historical background The rich heritage ancient medical literature of India makes no mention of kala-azar, the popular name of visceral leishmaniasis or any disease entity akin to it. Kala-azar is term coined in India and literally mean 'black fever' but could equally mean 'fatal fever'. The earliest available reference on the subject is contained in the annual report of the Inspector General of civil hospitals for 1872, in which he has quoted the civil surgeon of Burdwan district about the disease. The source from which kala-azar was introduced in Indian subcontinent remains a matter of conjecture. The advent of the disease in Bengal coincides with periodic incursions and raids by the Portuguese to the area, and it is possible that they introduced the disease either from their own country or from colonies from Africa. Alternative source could have been linkage with China through British maritime traffic. However, the disease in India is not identical with the disease in any of these areas, the nearest resemblance being with the East African form. Once introduced, the condition being appropriate, the disease possible adapted itself and then established a firm foothold Current situation in India In the Indian subcontinent (Bangladesh, Nepal and India), the most common endemic form of the disease is visceral leishmaniasis (VL) or kala-azar or Dum-Dum fever. Kala-azar is present in India for more than 100 years. The first appearance of kala-azar in India was recorded in 1862, when about 75, 000 cases were reported from Mohammadapur in Jessor district of East Bengal (now in Bangladesh) (Sen Gupta, 1944; Peters and Prasad, 1983). All the districts north of the river Ganges were affected with Kala-azar. Now the disease has spread southwards up to Darjeeling, MaIda, West Dinajpur and Burdwan districts of West Bengal bordering Bihar state. A sample survey in Bihar carried out in 1977, on the epidemic of kala-azar showed an estimated number of 100,000 cases in the state with 4500 deaths (Sanyal et ai., 1979); in 1989, 30,000 cases and 450 deaths. In 1990 infected cases reached to 54,000 with 590 deaths and by 1991 the number of cases increased to 250,000 with 75,000 deaths (Thakur et al., 1993). It is obvious that the number of reported cases largely underestimated. Some local surveys revealed that the real prevalence of disease was five times more than what was reported. The situation is particularly grave in the state of Bihar, India, known as the "heartland of kala-azar". It has recently been posed a 6

6 Chapter I : Review of literature serious threat involving 38 out of 42 districts of Bihar state, 8 districts of West Bengal and 2 districts of eastern Uttar Pradesh (Guerin el ai., 2002). At present the disease is present in almost all districts of Bihar, four districts of Jharkhand, five districts of Uttar Pradesh and 10 districts of West Bengal, 40 out of total 54 districts in Bihar are badly affected with VL. The known endemic districts of kala-azar are located north of the river Ganges namely Muzaffarpur, Vaishali, Darbhanga, Samastipur, Madhubani, East Champaran, Sitamarhi, Begusarai, Saran, Saharsa and Purnea (Fig.l.2). S :k k 1' 0 Dal bhsrg/i. G IWYA Nawada M unger. Jh... ~hard Fig.1.2: Map of resistance levels in the Bihar focus of visceral leishmaniasis, India. Orange-high-Ievel resistance, yellow-mixed resistance, green-no resistance (Trop Med Int Health 2002; 7: 293). Sporadic cases have also been reported from Gujarat (Gajwani el ai., 1967), Kashmir (Jacob and Kalra, 1951) Himachal Pradesh (Gupta and Bhatia, 1975). In Uttar Pradesh occurrence of sporadic cases of kala-azar started in the year 1987 with most of the cases reported so far from this state are imported cases (Thakur et ai., 1999) and in West Bengal 9 districts are affected including MaIda, Dinajpur and Darjeeling districts. Kala-azar has spread from Bihar to West Bengal, eastern Bangladesh, and eastern Uttar Pradesh and in the northern Nepal. In 2005 the health ministers of three Member States of WHO South-East Asia Region, India, Nepal and Bangladesh, had signed a Memorandum of Understanding pledging to collaborate to eliminate V L from their countries. 7

7 Geographical distribution of kala-azar closely coincides with the distribution of insect vector, Phlebotomus argentipes and ecological factors (Napier and Smith, 1926) (Shivaramakrishnamaiah and Ramanathan, 1967) such as: a) an altitude less than 2000 feet b) abundant rainfall more than 80 cm. annually and mean humidity of about 70% to 80% c) alluvial soil d) temperature below 38 C and above 4 C with diurnal variation less than 10 C e) abundant vegetation with subsoil water t) rural setting. All these conditions prevail in Assam valley, West Bengal, Tamil Nadu and Bihar (lyer, 1985) Disease and its epidemiology Leishmaniasis is not a single disease but a variety of syndromes that differ remarkably with one another. The WHO considers leishmaniasis as one of the most important parasitic diseases (WHO, 1990) Various forms of Leishmaniasis Leishmaniasis is a group of diseases with wide epidemiological and clinical diversity. The leishmaniasis is caused by over 20 species, pathogenic for humans, belonging to the genus Leishmania, a protozoa transmitted by the bite of a haematophagous insect vector, the phlebotomine sand fly. Governed by parasite and host factors and immunoinflammatory responses, the clinical spectrum of leishmaniasis encompasses subclinical (unapparent), localised (skin lesions), and disseminated infection (cutaneous, mucosal, or visceral). These wide-ranging differences of clinical manifestations define Leishmania virulence (degree of pathogenicity) in human infection. According to the form of the disease, site of infection and species involved, the leishmaniasis can be divided into following general clinical patterns Cutaneous Leishmaniasis (CL) CL is commonly known as oriental sore. Its causative agents are Leishmania major, L. tropica, L. aethiopica, L infantum in old World and L. mexicana, L. venezuelensis, L. amazonensis, L. braziliensis, L. panamensis, L. guyanensis, L. peruviana and L. chagasi are in New world. It produces skin lesions mainly on the face, arms and legs. It is frequently 8

8 Chapter I : Review of literature self-healing in the Old World but when the lesions are multiple and disabling with disfiguring scars, it creates a lifelong aesthetic stigma. Its most severe form, recidivans leishmaniasis, is very difficult to treat, long lasting, destructive and disfiguring. After recovery or successful treatment, cutaneous leishmaniasis induces immunity to re-infection by the species of Leishmania that cause the disease. Most commonly, it is prevalent in Mediterranean Basin, Syria, Arabia, and Mesopotamia, Persia to Central Asia, Central Africa and some parts of West em India Diffuse cutaneous Leishmaniasis (DCL) It is difficult to treat DCL due to disseminated lesions that resemble leprosy and do not heal spontaneously. This form is especially related to a defective immune system and it is often characterized by relapses after treatment Mucocutaneous Leishmaniasis (MCL) It is also called 'espundia' in South America. Causative Agents of MCL in Old World are Leishmania aethiopica (rare), L. major and in New World are L. mexicana, L. amazonensis, L. braziliensis, L. guyanensis and L. panamensis. The parasite invades the mucocutaneous region of the body and spread to the or,onasal/pharyngeal mucosa. The soft tissues and cartilage of the oronasal/pharyngeal cavity undergo progressive erosion. In contrast to cutaneous leishmaniasis, these lesions do not heal spontaneously. Suffering and mutilation are severe and death occurs as a result of bronchopneumonia or malnutrition. There is always a large danger of bacteria infecting the already open sores. Reconstructive surgery of deformities is an important part of therapy Visceral Leishmaniasis (VL) Visceral leishmaniasi:s (VL, kala-azar) is prevalent in 62 countries with an estimated annual incidence of 500,000. In India, the State of Bihar and adjoining areas of West Bengal, Jharkhand and Uttar Pradesh account for about half the world's burden of VL (Sundar et al., 2006). It is also known as 'Kala-azar' (in India). It is caused by Leishmania donovani complex i.e. L. donovani donovani (India, Africa), L. d. infantum (Middle East and some parts of Asia) and L. d. chagasi (South America). These species are morphologically indistinguishable but have been identified by molecular methods, predominantly multilocus enzyme electrophoresis. The disease can present an acute, subacute or chronic evolution, but most infected individuals remain completely asymptomatic (Bittencourt et al., 1995). The asymptomatic individual is characterized by positive serology to Leishmania and, possibly, a positive intradermal test. Infected 9

9 . individuals can evolve to a subclinical form of VL or directly to an overt form of disease (classical VL). Initially, the disease is characterized by high fever, headache, chill, malaise, dizziness, anorexia, and vomiting and weight loss. In chronic stage the disease is followed by hepatomegaly, splenomegaly, Iymphoadenopathy, occasional acute abdominal pain, emaciation, anemia, leucopenia, and blackness of skin, hence the name given kala-azar or Black fever. As the disease advances, splenomegaly can increase, causing abdominal distension and pain, which is sometimes increased by concomitant hepatomegaly followed by severe anemia and cachaxia. Symptoms and signs of bacterial co-infections such as pneumonia, diarrhea or tuberculosis can confuse the clinical picture at the time of initial diagnosis (Chappuis et al., 2007). It is the most severe form of leishmaniasis and is usually fatal (100% deaths) if left unattended. The incubation period can be months or years an<4 unlike the cutaneous forms of leishmaniasis, in this disease, the parasite uses the'" bloodstream to travel and it involves the internal organs such as liver, spleen, lymph nodes, and bone- marrow. After treatment and recovery, the patients may develop chronic cutaneous leishmaniasis that requires long and expensive treatment Post Kala-azar Dermal Leishmaniasis (PKDL) Post kala-azar dermal leishmaniasis is a sequel to the infection with L. donovani. Its causative agents in Old World are L. infantum, L. donovani, and L. tropica (rare; also may produce the atypical viscerotropic disease) and in New World L. chagasi is responsible for this. It is a type of non-ulcerative cutaneous lesion, developed in about 5 to 15 percent of kala-azar patients generally one or two years after completion of antimonial treatment (Rees and Kagar, 1987; Salotra et al., 2006). PKDL in India resembles lepromatous leprosy with verrucous papilomatous, xanthomathous and gigantic nodular forms; while in East Africa it resembles more to sarcoidosis and tuberculosis with popular rash over face or well defined rounded papules (Rashid et al., 1986) Epidemiology of Leishmaniasis It has been reported, there are an estimated 500,000 new cases for VL and more than 50,000 deaths from the disease each year (Desjeux, 2004), a death toll that is surpassed among the parasitic diseases only by malaria (WHO, 2002). Both figures are approximations as VL is frequently not recognized or not reported (Collin et al., 2006; Singh et at., 2006). The majority (>90%) of cases occur in just in six countries- Bangladesh, India, Nepal, Sudan, Ethiopia, and Brazil. Severe VL epidemics have been reported in the 10

10 Chapter I : Review of literature past in southern Sudan, in context of civil war and famine, VL killed and estimated 100,000 people out ofa population of 280,000 between (Jacquet et at., 2006). As India, Nepal and Bangladesh harbour an estimated 67% of the global VL disease burden (Hotez et at., 2004), the commitment of the government of these countries to launch regional VL elimination programme is welcome. The target of this programme is to eliminate VL as a public health problem by 2015, by using a local approach to reduce the annual incidence of. VL to less than one case per ten thousand individuals. Leishmaniases cause considerable morbidity and mortality. Leishmaniasis is a typical example of an anthropozoonosis. The majority of infections are originally zoonotic, although some cases are known of transmission of L. donovani from human to human. The different epidemiological cycles are: (i) a primitive or sylvatic cycle (human infection is accidental, transmission occurring in wild foci), e.g. L. braziliensis; (ii) a secondary or peridomestic cycle (the reservoir is a peridomestic or domestic animal, the parasite being transmitted to humans by anthropophilic sand flies), e.g. L. infantum; and (iii) a tertiary, strictly anthroponotic cycle (in which the animal reservoir has disappeared, or not yet been identified, and the sand fly vectors are totally anthroponotic) e.g. L. donovani. Nevertheless, many unknown factors remain. For example, the main animal reservoir of L. braziliensis is still unknown (Cupolillo et at., 2003). L. tropica was considered to be a strict anthroponosis, but several cases of canine infection have been described (Dereure et at., 1991 a; Dereure et al., 1991 b; Yahia et at., 2004). The epidemiology of leishmaniasis in a given area is directly dependent on the behavior of the human and/or animal population in relation to the cycle of transmission. There are a variety of factors that influence the transmission of the disease (Lane, 1993; Kettle, 1995). They are as follows: Proximity of residence to sand fly breeding and resting sites Type of housing Occupation Extent of exposure to sand fly bites Natural resistance, genetic or acquired Virulence of the parasite species Zoonotic or anthroponotic reservoirs. It seems that zoonotic reservoirs are particularly stable when wild uncontrolled populations (e.g. rodents) are involved. Up till now it has been observed that humans are not a reliable agent because of death and treatments except 11

11 Chapter I : Review of literature of the chronic condition of PKDL. Nevertheless recent reports about asymptomatic infections in healthy blood donors in France (Ie Fichoux et al., 1999) are adding a new parameter to the later The vectorial capacity, which is defined as the number of infective bites delivered per human per annum (Dye, 1992) Density, seasonality, longevity and flight range of sandfly populations Anthropophilia or zoophilia of sandflies and degree of it Entomology The insect vectors of Leishmania parasites are sandflies belonging to the family Psychodidae, sub- family Phlebotominae and genera Phlebotomus (Old World) and Lutzomyia (New World) with hundreds of species spread all over the world (Volf et al., 1994). Of the 500 known phlebotomine species, only 30 of them have been positively identified as vectors of the disease. Only the female sandfly transmits the protozoa, infecting itself with the Leishmania parasites contained in the blood, during the blood meal from human or mammalian host in order to obtain the protein necessary to develop its eggs. In the Old World (Europe, Asia, and Africa) sandfly vectors belong to the genus Phlebotomus and in the New World (America), to the genera Lutzomyia and Psychodopygus. The gut of the haematophagous insect is a potentially nutrient rich but highly specialized environmental niche, and the successful development of ingested potential pathogens or parasites such as Leishmania depends on their ability to avoid or adapt to the dramatic changes in the physicochemical environment accompanying bloodmeal and sugar-meal digestion. The strategy of the mosquito-borne malaria parasite is to exit rapidly through the gut epithelium and continue development in the hemocoel. In contrast, African trypanosomes in tsetse flies (Aksoy et al., 2004) and Leishmania (Sacks and Kamhawi, 2001) have adapted to remaining and developing in the insect gut. The Leishmania parasite is supremely adapted to the gut environment of the sand fly, secreting a unique gel like material composed mainly of a high-molecular-weight filamentous proteophosphoglycan (f PPG). Leishmania fppg serves a dual function, first blocking the fly gut and improving chances for transmission and subsequently aiding survival of the parasite in the mammalian host (Rogers et ai., 2002; Warner et al., 2004). There are different vectors in different regions for a single spp, for example, vectors of Leishmania donovani are Phlebotomus argentipes in India, P. chinensis in China, P. perniciosus in North Africa, Italy, France and Portugal, P. perfiliewi in Greece, P. orietalis 12

12 in Sudan, and Ethiopia, P. martini in Kenya (Le B1ancq and Peters, 1986), and vectors L. in/antum are P. erniciosus, P. ariasi, P. perfiliewi and P. neglectus. Sometimes a single species is transmitted by a single vector, e.g. L. chagasi is transmitted by sandflies belonging to the genus Lutzomyia (L. longipalps) (WHO, 1990). Transmission of parasite may be anthroponotic (from one human to another) or zoonotic (from animal to human). In India, the disease is completely anthroponotic where as in certain parts of the world, there are one or more reservoirs (zoonotic host) e.g. dogs in the Mediterranean region and rodents in South Africa Distribution of vector in India Phlebotomus argentipes prefers Yhot and humid climates in all the VL abundant endemic areas of Bihar, West Bengal, Assam and Eastern Utter-Pradesh. High densities have also been recorded in Southern peninsula and Central India. Vertical distribution has, been reported to up to 1300 m above sea level in Garhwal (Utteranchal) and 1100 m in Nilgiri Hills (Tamil Nadu) Habit and habitats The vector is crepuscular in its habit, inactive dudng daytime, and seeks shelter in err cracks and crevices in the dark corners of houses 96 cattlesheds (Pal it et at., 1996). In outdoor situations, it is found in caves, crevices, animal burrows, termite hills, tree holes etc. The sandflies are incapable of flying long distances and move by characteristic hopping movement. They have been detected up to a height of 2.74 m from the ground. It is found throughout the World inter tropical and temperate regions Seasonal prevalence Studies conducted in endemic areas revealed that the vector density starts increasing from February onwards, with some decrease in May to June, followed by an increase with the advent of the monsoon. In Southern and Eastern India, with very mild cold season, P. argentipes is common throughout the year VL control strategies The current control strategies for VL rely on reservoir and vector control, the use of insecticide-impregnated materials and active case detection and treatment (Boelaert et at., 2000; Davies et at., 2003) anti-leishmanial vaccines are still being developed. 13

13 Chapter I : Review of literature Reservoir control Dogs are the main reservoir of L. infantum in zoonotic VL. Despite evidence from experimental studies showing a decreased incidence of VL in both dogs and children following serological screening of dogs and killing of sero-positive animals (Ashford et ai., 1998; Palatnik-de-Sousa et al., 2001), the efficiency and acceptability of this control strategy is increasingly being debated (Alvar et al., 1994; Tesh, 1995; Reithinger and Davies, 2002). Treating infected dogs is not an effective control strategy as relapses are frequent and dogs can regain infectivity weeks after treatment, despite being clinically cured (Alvar et al., 1994). Moreover, the widespread veterinary use of VL drugs might lead to resistance in parasites. A new control approach is the use of deltamethrine-treated collars, which reduced the risk of infection in dogs (by 54%) and children (by 43%) in a study conducted in Iran (Gavgani et al., 2002). Vaccination of dogs would nevertheless be the best strategy if an efficacious vaccine can be developed Vector control Sandflies are susceptible to the same insecticides as Anopheles mosquitoes, the malaria vector. Residual insecticide spraying of houses and animal shelters was shown to be efficacious in India (Kaul et al., 1994), where the vector (Phlebotomus argentipes) is restricted to areas in and around the home. Following the large scale antimalarial insecticide (dichloro-diphenyl-trichloroethane (DDT)) spraying campaigns that were implemented in the 1950s, VL almost completely disappeared from the Indian subcontinent. Unfortunately, the disease quickly re-emerged when these spraying campaigns were discontinued. Resistance of P. argentipes to DDT remains limited, but has been reported in Bihar (Singh et al., 2001). In Sudan and other endemic countries in East Africa, transmission occurs mainly, but not exclusively (Hassan et ai., 2004), outside villages, during shepherding for example. Indoor residual spraying for disease control is therefore unlikely to be as efficient in this region Insecticide-impregnated materials The use of insecticide- treated bednets (ITNs) could concomitantly prevent VL and other vector-borne diseases, such as malaria and Japanese encephalitis. There is limited evidence that bednets provide protection against VL. Case-control studies conducted in Bangladesh and Nepal showed that sleeping under a non-impregnated bednet during the warm months was a protective factor against VL (Bern et al., 2000; Bern et al., 2005). Despite low usage, the mass distribution of ITNs in Sudan was accompanied by a 27% 14

14 Chapter I : Review of literature reduction in the incidence of VL in an observational study (Ritmeijer et al., 2007). A large prospective randomized controlled trial testing the efficacy of long-lasting ITNs to prevent L. donovani infection and VL is underway in Nepal and India. Depending on the sleeping traditions of the population and the biting habits of the local vector, other insecticideimpregnated materials such as curtains and blankets should be evaluated for use in VL prevention, as some have been shown to provide efficient protection against cutaneous leishmaniasis (Reyburn et al., 2000; Kroeger et al., 2002) Life cycle During their complex life cycle, the singled cell parasites of the genus Leishmania are exposed to different extra and intracellular environments. These organisms are digenetic parasites with two basic life cycle stages: one extracellular stage within an invertebrate host (phlebotomine sand fly) and one intracellular stage within a vertebrate host. Thus, the parasites exist in two main morphological forms, amastigotes and promastigotes, which are found in vertebrate hosts and invertebrate hosts, respectively (Fig.I.3). 15

15 Chapter I : Review of literature Intracellular amastigote Prol iferation r Phagolysosome Uptake A'~'C""'" Sandfly bill! - -"" AmaStigotesg g P> <'/C';' \ / promastigotes d~ Proliferation in the midgut /' Fig.1.3: Life cycle of Leishmania parasite (Source CDC 2004, 53( 12); ) Stages in the Invertebrate Host (Promastigote) The invertebrate hosts or vectors are small insects of the order Diptera, belonging to the subfamily Phlebotominae. They are commonly called phlebotomine sand flies. Of the six genera described, only two are of medical importance: Phlebotomus of the ' Old World', divided into 12 subgenera, and Lutzomyia of the 'New World', divided into 25 subgenera and species groups. All known vectors of the leishmaniases are species of these two genera. Among the 500 known phlebotomine species, only 31 have been positively identified as vectors of pathogenic species of Leishmania and 43 as probable vectors (Volf et al., 1994; V Killick-Kendrick, 1999). The sand fl y species involved in the transmission of Leishmania 16

16 vary from one geographical region to another but also depend on the species of Leishmania (Volf et al., 1994; Killick-Kendrick, 1999). Like mosquitoes, the female needs a blood meal for egg development and only the female is haematophagous. Some phlebotomine species can support the growth of only those species of Leishmania with which they are infected in nature, such as Phlebotomus papatasi and P. sergenti; these species are considered to be restricted vectors (Kamhawi et al., 2000). By contrast, other phlebotomine species such as Lutzomyia longipalpis and Phlebotomus argentipes are permissive vectors since they are able to develop mature transmissible infections when infected with several Leishmania species (Kamhawi et al., 2000; Sadlova et al., 2003; Warner et al., 2004). Within the intermediate host, Leishmania develops as promastigote forms with a cell body measuring 5-20 x l-4mm, elongated motile extracellular stages possessing a prominent free flagellum up to 20 mm-iong. Nevertheless, a variety of different promastigote forms have been distinguished on morphological grounds (Bates and Rogers, 2004) Stages in the Vertebrate Host (Amastigote) In the vertebrate host, the parasite evolves into an amastigote form. Amastigotes are ovoid (2.5-5 mm diameter), non-motile, intracellular stages. They do not have a free flagellum and are located in the parasitophorous vacuoles of the host's macrophages. In both developmental forms, the flagellum emerges from a flagellar pocket and in the amastigote form, it is almost completely restricted to it, so it is only observed by electron microscopy. Infection begins when an infected female sand fly takes a bl60d meal from a healthy human host. Following inoculation into the skin by the sand fly bite, the infective flagellated metacyclic promastigotes are ultimately ingested by macro phages via receptormediated endocytosis (Chang et al., 1990), transforms into amastigotes, and multiplies by binary fission. The infected macrophage eventually bursts and the released parasites are able to infect new phagocytic cells. When the infected host is bitten by another female sand fly, at the time of infective blood meal, the amastigotes in the gut of sandfly, due to change in temperature and other conditions, develop in to flagellated promastigotes and the life cycle continues Current options for treatment Despite the considerable progress made in the study of the biochemistry, physiology and molecular biology of Leishmania parasites, the absence of effective vaccines and vector 17

17 control programs, makes chemotherapy the only tool against leishmaniasis. The current situation for the chemotherapy of leishmaniasis is more promising than it has been for several years with both new drugs and new formulations of old drugs either recently approved or on clinical trial (Guerin et al., 2002; Croft and Coombs, 2003; Croft et al., 2006). The drugs available for treatment of Leishmania infections are as under Antimonials Antimony (Sb) was acclaimed as one of the "Seven Wonders" of the World published in a treatise in Leipzig in It was introduced by Paracelsus as a general panacea in the 16 th century, occasionally banned and often argued over for another three centuries, the modern era of usage began in 1905 when Plimmer and Thompson showed the activities of sodium and potassium tartrate (PTA) against trypanosomes in rats and subsequently in the treatment of human trypanosomiasis in Africa. The first published records of use of these trivalent antimonials for treatment were by Macado and Vianna in 1913 for CL, by di Cristina and Cariona in Sicily and Rogers in India in 1915 for VL (Schmidt and Peter, 1938). The discovery and synthesis of urea stibamine in 1920, by Prof. U.N. Brahmachari, was the most significant milestone in the control ofvl. The death of Sir Brahmachari in 1946 and the dramatic decline in the incidence of Kala-azar due to destruction of Phlebotomus argentipes, the common vector of L. donovani in India, by DDT used to malaria destruction programme after World War II resulted in misuse of urea stibamine. Its success became overshadowed and paved the way of the development of the less toxic pentavalent antimonials. In 1937 Brahmachari, Schmidt, Kikuth and others led to the synthesis of antimony gluconate (Solustibosan) (Kikuth and Schmidt, 1937) and sodium stibogluconate (Pentostam) in 1945 (Goodwin, 1995). Another carbohydrate complex, meglumine antimoniate (Glucantime. Aventis) soon followed. Therefore, important antimonial drugs are tartar emetic, sodium stibogluconate, meglumine antimoniate and derivatives of stibanilic acid such as stibamines, stibacetin, ethyl stibamine and urea stibamine. Out of these drugs sodium stibogluconate and meglumine antimoniate were found to be highly effective. Although, the structure of stibogluconate is still unknown despite its use for 50 years as the first line drug for VL. Treatment of leishmaniasis still relies to a large extent on pentavalent antimony compounds such as meglumine antimoniate (Glucantime, Aventis) and sodium stibogluconate (Pentostam, GSK) (Herwaldt, 1999; Guerin et ai., 2002). 18

18 (a) Sodium stibogluconate- This is the safest and most potent drug among the pentavalent antimonials and has been widely used for nearly a half century (Cook, 1990) to treat VL rather than cutaneous and mucocutaneous leishmaniasis (Thakur et al., 1988) finding wider usage in India, China, Middle East, Africa and North America. The WHO (WHO, 1990) has recommended a daily dose of Sb v 20 mg/kg for 30 days. In India, up to 60% VL patients in Bihar state do not respond to pentavalent antimonials (Croft and Coombs, 2003). An altered regimen of 20 mg/kg per day has been recommended for more than 40 days (Thakur et al., 1988). Bryceson, (1987) proved that Sb v is more effective if given more frequently than once daily and his data was corroborated by data of Zij Istra et al. (1991) who showed that Sb v at 10 mg/kg every 12h for 15 d was better than 20 mg/kg per day for 30 days. Chulay et al. (1983) also showed that 10 mg/kg every 8h for 10 days was more effective than 20 mg/kg per day for 30 days. (b) MegIumine antimoniate- This is also a drug of choice for treating leishmaniasis and has been widely used in Latin America at the recommended adult dose of mg/kg/d for 10 to 20 days. Although pentavalent antimony compounds have proven to be very effective, drug use is often limited in patients due to toxic side effects such as nausea, abdominal pain, chemical pancreatitis, renal toxicity and, especially worrisome, electrocardiographic abnormalities (Guerin et al., 2002). The cardiotoxity of pentavalent antimony compounds, which may include inversion of the ST-segment on the electrocardiogram, QTc prolongation, torsade de pointes arrhythmias (TdP) and sudden cardiac arrest (Chulay et al., 1985; Ortega-Carnicer et al., 1997; Thakur, 1998; Berhe et al., 2001) (Cesur et al., 2002), severely limits prolonged treatment courses in patients in particular when high concentrations are indicated to combat and overcome resistance. The exorbitant cost of brand formulations of Sb v prompted Medicins Sans Frontieres to commission three studies in Sudan, Kenya and Ethiopia to compare the efficacy in VL of the generic sodium antimony gluconate (SAG) (Albert David, Kolkata India, costs US $13 per patient) vs. branded SAG (Pentostam, Glaxo- Wellcome, UK, costs US $200 per patient) (Veeken and Ritmeijer, 2006). It was conclusively proven that no significant difference existed between the two formulations as generic SAG was equally effective in terms of efficacy and safety in all forms of leishmaniasis and importantly, 19

19 achievable at a substantially lower cost. However, caution must be exercised before using Sb v from new manufacturers as bad batches caused fatal cardiotoxicity (Sundar et al., 1998). In two reports from India and Nepal, high incidence of fatal cardiotoxicity was reported with use of antimony made from an unknown manufacturer (Sundar et al., 1998; Rijal et al., 2003). Post kala-azar dermal leishmaniasis (PKDL), a dermatological manifestation generally following VL infection occurs predominantly in India and Sudan. Although in both L. donovani is the causative organism, Indian PKDL requires prolonged treatment (> 120 days) (Thakur and Kumar, 1990) whereas for the Sudanese variety, two months treatment is considered adequate (Kamil et ai., 2003). j The precise mechanism of action remains unknown, although antimonial compounds have been used in the treatment of leishmaniasis for at least 100 years. Interpretation of some of the earlier reports on mode of action and drug sensitivity to antimonials is complicated by the fact that liquid formulations of sodium stibogluconate contain the preservative m-chlorocresol, itself a potent antileishmanial agent (Brown et al., 1993). However, Goodwin and Page (1943) were the first to propose that pentavalent antimony Sb v might act as a prodrug that has to be converted into active, trivalent Sb III which is thought to interfere with thiol-dependent redox systems of Leishmania parasites (Kellinghaus et ai., 2004). The details of the activation mechanism and the exact site of this conversion of Sb v to Sb III are still unclear. However, several studies have reported that axenic amastigotes (i.e., culture turned in the absence of macrophages) are susceptible to Sb v, whereas promastigotes are not, suggesting that some stage specific reduction occurs in this life cycle stage (Ephros et ai., 1999; Goyard et al., 2003). Biochemical studies over the past two decades have indicated a number of potential targets for pentavalent antimonials; glycolysis in particular inhibition of ADP phosphorylation (Berman et ai., 1985), DNA I topoisomerase (Chakraborty and Majumder, 1988; Lucumi et al., 1998), inhibition of fatty acid beta-oxidation (Berman et al., 1989) and trypanothione (Mukhopadhyay et al., 1996; Legare et ai., 1997). Recent studies have shown that both Sb III and Sb v mediate DNA fragmentation in Leishmania species, suggesting that antimony kills the parasite by a process reminiscent of apoptosis (Sereno et al., 200 I a; Lee et al., 2002; Sudhandiran and Shaha, 2003). / 20

20 / TH16417 Chapter I : Review of literature Pentamidine Pentamidine, an aromatic diamidine, as the isethionate salt (Pentacarinat) and previously as the methyl sulphonate salt (Lomidine), have been used as alternative treatments for VL, CL and DCL since As a second line drug for antimony resistant cases it has proved useful in India and Kenya (Thakur et ai., 2001). The efficacy of pentamidine against antimony refractory infections has decreased over the years (Jha et ai., 1991; Sundar, 2001). Its use has been largely abandoned in India where pentamidine failures are common, but it continues to be used alone or in combination with other drugs in other countries (Basselin et al., 1997a; Heath et al., 200 I). Although use of pentamidine for the treatment of CL was revisited in the 1990s, with clinical trials for treatment of New World CL, this drug is not a widely used antileishmanial drug. Pentamidine was shown to be highly effective against CL in Colombia in a short course low dose regimen (Soto et al., 1994). For VL there are some indication of emergence of resistance have been reported. Pentamidine resistant promastigote clones of L. donovani and L. amazonensis (Basselin et al., 2002). Although, specific transporters for pentamidine uptake have been characterized and might have a role in resistance (Bray et al., 2003; Coelho et al., 2003). Coelho et al. (2003) have reported pentamidine resistant gene belonging to the P-glycoprotein (PGP)IMRP ATP-binding cassette (ABC) transporter superfamily, named pentamidine resistance protein 1 (PRPl), might have a role in pentamidine resistance. Others data have shown the accumulation of more pentamidine in the wild type Leishmania mitochondrion than the resistant cells, is being of importance for more drug efflux (Basselin et at., 2002). The antileishmanial mechanism of action of pentamidine remains incompletely understood (Bray et ai., 2003), and there are probably multiple targets, including polyamine biosynthesis (Basselin et al., 1997b), DNA minor groove binding and mitochondrial inner membrane potential (Vercesi and Docampo, 1992). Toxicity has always been a limitation on use with reports of hypoglycaemia, diabetes, nephrotoxicity, tachycardia, pain at site of injection (Jha, 1983; Soto et al., 1994). Pentamidine is still used for treatment of haemolymphatic stage of human African trypanosomiasis (Pepin and Milord, 1994) and, in combination with sulfamethoxazole, for Pneumocytis carinii pneumonitis (PCP) in AIDS patients Amphotericin B and its lipid formulations Amphotericin B (Amp B), a macrolide polyene antibiotic isolated from Streptomyces nodosus, was first shown to have antileishmanial activity in the early 1960's? )~. :; 9 IH,- J 64( R1451 Ch TH

21 and was soon used in the treatment of mucocutaneous leishmaniasis (Sampaio et ai., 1960; Fuchs et al., 2003). Amp B is predominantly used as an antifungal drug, specifically for treating systemic mycoses. The selective activity of Amp B against fungi and Leishmania is due to the higher affinity of the drug for 24-substituted sterols, found in the plasma membrane of these eukaryotic microorganisms, over cholesterol in the plasma membranes of mammalian cells. Amp B in a dose of mg/kg for 15 to 20 infusions either daily or on alternate days has consistently cured about 97 per cent of VL patients (Sundar and Rai, 2002). Although Amp B has long been considered as an alternative treatment for MCL and VL (WHO, 1996), its use has been restricted by infusion related and delayed toxic side effects, in particular cardiotoxicity and nephrotoxicity and even deaths (Khoo et al., 1994). However, antileishmanial chemotherapy has benefited from the development of lipid-associated formulations of Amp B. In these formulations, deoxycholate has been replaced by other lipids that mask toxicity of Amp B and facilitate its preferential uptake by reticuloendothelial cells, thus achieving targeted drug delivery to the parasite resulting in increasing efficacy and reduced toxicity and an extended plasma half-life in comparison to the parent drug. Three such lipid associated formulations are: (i) Liposomal amphotericin B (AmBisome; Gilead Sciences, Foster City, CA,USA) (U) Amphotericin B lipid complex (Abelcet; The Liposome Co, Princeton, NJ, USA)and (iii) Amphotericin B colloidal dispersion (Amphocil; Sequus Pharmaceutical; Menlo Park, USA). These formulations have been successfully used in clinical trials for VL and/or MCL while, AmBisome was proven quite effective (Berman et al., 1987). In Bihar (India) a comparative study of parental Amp B (1 mg/kg x 30, at alternative days) with AmBisome and Abelcet (both at a dose of2 mg/kg/day for 5 days) showed the similar overall cure rates but, the lipid formulations had an upper edge as they produced distinctly lower toxicities. Liposomal Amp B is considered by many experts as the best existing drug against VL, and is used as first-line treatment in Europe and the United States However, in terms of cost factor, the lipid formulations were two times more expensive than Amp B (Sundar et ai., 2004). Until recently, its use in developing countries was precluded by its high market price (US$2,800 per treatment) (Gradoni et al., 1993; Bern et al., 2006). This situation might change as the World Health Organization (WHO) announced a drastic price reduction in 22

22 Chapter I : Review ofliterature May 2007, with the cost of an average course for the public health sector in VL-endemic countries reduced to US$200. There have been two small inconclusive studies on the emergence of Amp B resistance in L. infantumlhiv -infected cases in France. One study failed to find a change in sensitivity in promastigotes derived from isolates taken before and after the treatment of one patient (Durand et ai., 1998). In contrast, a decrease in sensitivity was observed in isolates taken over several relapses from another patient (Di Giorgio et al., 1999). There has been increased use of Amp B for visceral leishmaniasis, in both the deoxycholate (Thakur et ai., 1998; Sundar et al., 2002b) and lipid formulations (Berman et al., 1998; Sundar et al., 2003), following failure of antimonial treatment and in HIYNL coinfection cases. With the increasing use of amphotericin B in lipid formulations that have longer half-lives, the possibility of resistance cannot be ignored Paromomycin (aminosidine) Paromomycin (PM), an aminoglycoside antibiotic, was originally identified as an antileishmanial in the 1960s and has been used for the treatment of VL in parenteral formulation in phase III and CL in both topical and parenteral formulations. Although development of the parenteral formulation of PM, a drug with poor oral bioavailability, for VL has been slow, several Phase 2 trials in India and Kenya have been promising, with 90% of patients cured ofyl. There are also encouraging findings on the use of PM as a topical treatment for CL. Elon and colleagues in 1984 have reported that a topical formulation containing 15% PM and 12% methyl benzethonium chloride (a skin penetrating agent) was effective against experimental CL led to clinical trials. Hydrophillic formulation of PM was found more effective than hydrophobic formulation (Goncalves et al., 2005). Combinations of paromomycin with antimonials have been effective against YL (Nuzum et al., 1995; Thakur et al., 2000) with the potential shortening of treatment. It acts by interfering with the mitochondrial activity and inhibiting cell respiration and lowering the electric potential difference across the mitochondrial membranes (Maarouf et ai., 1998). Some studies indicated that binding to ribosomal units might be involved as paromomycin promoted subunit dissociation of both cytoplasmic and mitochondrial ribosomes (Maarouf et al., 1998). 23

23 The results from initial studies in India and Africa were promising (Chunge et ai., 1990; Jha et al., 1998), but the original manufacturer abandoned production. The results of Phase III trials recently conducted in India showed excellent efficacy and safety. No nephrotoxicity was observed; reversible high-tone ototoxicity (damage to the inner ear) was found in 2% of patients and 1.8% of patients showed a significant increase (> fivefold) in hepatic transaminases (Sundar et al., 2007). Mild injection pain was reported by over 50% of patients. Paromomycin was registered in India in August In East Africa, paromomycin is currently being evaluated in mono- and combination (with sodium stibogluconate) therapy. Other advantages of paromomycin include the fact that it is active against a wide variety of pathogens, including bacteria, and its low cost (US$5-10 per treatment). Resistance to aminoglycosides in bacteria is well known and has been characterized In relation to decreased uptake in gram-negative pathogens, alteration of the ribosomal binding, and modification of amino groups or hydroxyl groups by inactivating N- acetyltransferases, O-phosphotransferases, or O-nucleotidyl transferases (Davies and Wright, 1997). Paromomycin has had limited use in the treatment of visceral leishmaniasis, and there is no clinical resistance in this form of the disease have been reported. However, it has been used more extensively for the treatment of cutaneous disease. So far, there has been only one report suggesting resistance could develop. Following a 60-day parenteral course for treatment of two L. aethiopica cases, isolates taken from relapse patients were three- to fivefold less sensitive to the drug after treatment than isolates taken before treatment in an amastigote-macrophage assay (Teklemariam et ai., 1994). In studies on selected populations of promastigotes, resistance was related to decreased drug uptake in L. donovani (Maarouf et al., 1998). In the analysis of small-subunit rrna and DNA of a paromomycin-resistant L. tropica, small-subunit rrna was eightfold more resistant to paromomycin and had a low level of cross-resistance to other aminoglycosides, showed no change in the sequence of the binding site. Monitoring of resistance could be of importance if paromomycin formulations are introduced as a first line treatment (Croft et al., 2006) Allopurinol and purine analogues The antileishmanial activity of the purine analogue allopurinol was identified over 30 years ago and, because it had oral bioavailability and was widely used for other clinical indications, it entered clinical trials for VL and CL. All parasitic protozoa studied to date are unable to synthesize purines de novo and, therefore, rely upon uptake and salvage of 24